Korean J Physiol Pharmacol.  2008 Apr;12(2):51-58. 10.4196/kjpp.2008.12.2.51.

Expression of Ca2+ -activated K+ Channels and Their Role in Proliferation of Rat Cardiac Fibroblasts

• Choi S
• Lee W
• Yun J
• Seo J
• Lim I

• KMID: 1486143
• DOI: http://doi.org/10.4196/kjpp.2008.12.2.51

Abstract

Cardiac fibroblasts constitute one of the largest cell populations in the heart, and contribute to structural, biochemical, mechanical and electrical properties of the myocardium. Nonetheless, their cardiac functions, especially electrophysiological properties, have often been disregarded in studies. Ca2+-activated K+(KCa) channels can control Ca2+influx as well as a number of Ca2+-dependent physiological processes. We, therefore, attempted to identify and characterize KCa channels in rat Cardiac fibroblasts. First, we showed that the cells cultured from the rat ventricle were cardiac fibroblasts by immunostaining for discoidin domain receptor 2 (DDR-2), a specific fibroblast marker. Secondly, we detected the expression of various KCa channels by reverse transcription polymerase chain reaction (RT-PCR), and found all three family members of KCa channels, including large conductance KCa (BK-alpha 1- and -beta 1~4 subunits), intermediate conductance KCa (IK), and small conductance KCa (SK1~4 subunits) channels. Thirdly, we recorded BK, IK, and SK channels by whole cell mode patch clamp technique using their specific blockers. Finally, we performed cell proliferation assay to evaluate the effects of the channels on cell proliferation, and found that the inhibition of IK channel increased the cell proliferation. These results showed the existence of BK, IK, and SK channels in rat ventricular fibroblasts and involvement of IK channel in cell proliferation.

Keyword

Cardiac fibroblasts; Ca2+ -activated K+ channels; Proliferation

MeSH Terms

Animals
Cell Proliferation
Fibroblasts
Heart
Humans
Myocardium
Physiological Processes
Polymerase Chain Reaction
Rats
Receptor Protein-Tyrosine Kinases
Receptors, Mitogen
Reverse Transcription
Receptor Protein-Tyrosine Kinases
Receptors, Mitogen

Figure

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  Fig. 1. The expression of DDR-2 (a specific fibroblast marker) in rat cardiac fibroblast cells. Rat cardiac cells were double stained by DDR-2 antibody and PI. DDR-2 was positive also in fibroblast cell line (CRL-1474), but not skeletal myoblast cell line (CRL-1458).

• 

  Fig. 2. Identification of various KCa channels in rat cardiac fibroblasts by RT-PCR. The size of amplification products was BK-α1 (206 bp), BK-β1 (229 bp), BK-β2 (172 bp), BK-β3 (144 bp), BK-β4 (242 bp), IK (221 bp), SK1 (264), SK2 (145 bp), SK3 (179 bp), and SK4 (211 bp). GAPDH was used as positive control and the size of amplification products was 280 bp.

• 

  Fig. 3. Identification of BK, IK, and SK channels in rat ventricular fibroblasts by whole-cell mode patch clamp technique. Outward K+ currents were generated by incremental 10 mV depolarizing steps from −60 mV to 50 mV for 500 ms and the holding potential was −50 mV. (A) Representative effect of IBTX (0.1μM) on the outward K+ currents in fibroblast and the I-V curve shows the mean values of steady state currents. (B) Representative effect of clotrimazole (CLT, 2μM) and (C) apamin (0.5μM) on the K+ currents in other cells, and their I~V curves are shown. (D) The effects of various KCa channel blockers on the amplitude of the K+ currents of rat cardiac fibroblasts at 50 mV. Data show mean±S.E.M. of indicated numbers (∗p<0.05, ∗∗p<0.01, compared with 100% of control).

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  Fig. 4. Investigation for the expression of three types of KCa channels in a fibroblast of rat ventricle by whole-cell mode patch clamp technique. Currents were evoked by 500 ms step depolarizion to 70 mV from −40 mV. Holding potential was −50 mV. (A) The effects of various KCa channel blockers on the outward K+ currents in one fibroblast. Apamin (0.5μM), clotrimazole (CLT, 2μM), or IBTX (0.1μM) was added to the bath solution. (B) The I~V curve shows the mean values of steady state currents in the fibroblast.

• 

  Fig. 5. The effects of various KCa channel blockers on the proliferation of the rat ventricular fibroblasts. IBTX (0.1μM), clotrimazole (CLT, 2μM), and apamin (0.5μM) were tested. Only clotrimazole (2μM) significantly increased cell proliferation (∗∗p<0.01 from control at 24 hr). The control response was set as 100%, which is represented by optical density of unstimulated cells. Data show mean±S.E.M. of observations in 24 wells (2,500~3,000 cells/well).

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